Initiation of placental differentiation represents the earliest event in mammalian embryogenesis. Formation of the trophoblast epithelial structures of the placenta villi is critical to viability and function of the fetal-maternal unit. Defects in trophoblast cell expansion, terminal differentiation, and/or function underlie a wide spectrum of obstetric disorders. Early during development the embryonic trophectoderm gives rise to two distinct cytotrophoblast lineages: extravillous cytotrophoblasts that invade and modify maternal decidua to support the growing fetus and the villous syncytiotrophoblastic epithelium (STB). The STB layer of the placental villi comprises the interface between fetal and maternal circulations and serves the critical functions of nutrient and gas exchange and establishment of a maternal/fetal hormonal environment. The goal of this grant is to define molecular events central to the formation and function of the STB. We will use complementing cell culture and animal models of STB differentiation to focus on the pathway(s) involved in activation of STB-specific genes. Activation of the chorionic somatomammotropins (hCS) and growth hormone variant (hGH-V) genes of the human growth hormone gene (hGH) cluster will be used as paradigms for STB gene expression. Expression of these genes is tightly linked to STB differentiation and by the second half of pregnancy these genes encode the major pregnancy hormones of maternal serum. Emphasis will be placed on defining epigenetic pathways and chromatin modifications leading to hCS and hGH-V transcriptional activation. Models based on these data will be tested in developmentally dynamic settings of ex vivo-differentiating trophoblast cells and in the transgenic mouse placenta. Four Specific Aims are proposed: Aim I. Define the progression of epigenetic chromatin modifications at the hGH gene cluster in differentiating STB, Aim II. Establish linkages between STB differentiation and hCS gene expression, Aim III. Characterize the role of the P-element in activation of placental hormone expression from the hGH cluster, and Aim IV. Map long-range interactions between the hGH LCR and hGH cluster in STB chromatin that specify placental gene activation. These studies will contribute to the understanding of placental differentiation and function and should contribute to diagnostic and therapeutic advances in the field of maternal and fetal health.